Textbook Question
Describe the various characteristics of the Watson–Crick double-helix model for DNA.
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7. DNA and Chromosome Structure
DNA as the Genetic Material
2:39 minutesProblem 22
Textbook Question
What is the hyperchromic effect? How is it measured? What does Tₘ imply?
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The hyperchromic effect refers to the increase in the absorbance of ultraviolet (UV) light by DNA when its double-stranded structure is disrupted, such as during denaturation. This occurs because the nitrogenous bases in DNA, which are normally stacked and shielded in the double helix, become exposed and absorb more UV light when the strands separate.
To measure the hyperchromic effect, a spectrophotometer is used to monitor the absorbance of UV light at a wavelength of 260 nm (the peak absorbance for DNA). The absorbance is recorded as the DNA sample is gradually heated, causing the double-stranded DNA to denature into single strands.
The melting temperature (Tₘ) is the temperature at which half of the DNA molecules in the sample are denatured, meaning 50% of the double-stranded DNA has separated into single strands. This is observed as the midpoint of the sharp increase in absorbance during the heating process.
Tₘ provides important information about the stability of the DNA double helix. Factors such as the GC content (guanine-cytosine base pairs) and the ionic strength of the solution influence Tₘ, as GC pairs have three hydrogen bonds and contribute more to stability than AT pairs, which have two hydrogen bonds.
To determine Tₘ experimentally, plot a graph of absorbance at 260 nm versus temperature. The Tₘ is identified as the temperature corresponding to the midpoint of the sigmoidal curve that represents the DNA melting process.
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Key Concepts
Here are the essential concepts you must grasp in order to answer the question correctly.
Hyperchromic Effect
The hyperchromic effect refers to the increase in absorbance of ultraviolet (UV) light by nucleic acids, particularly DNA, when they are denatured or separated into single strands. This phenomenon occurs because the bases of DNA are more exposed and accessible to UV light when the double helix unwinds, leading to a higher absorbance at 260 nm. It is a crucial concept in molecular biology for studying DNA stability and interactions.
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Measurement of Hyperchromic Effect
The hyperchromic effect is typically measured using UV-Vis spectrophotometry, where the absorbance of a DNA sample is recorded before and after denaturation. By comparing the absorbance values, researchers can quantify the extent of denaturation and infer the stability of the DNA structure. This measurement is essential for understanding the thermal properties of nucleic acids and their interactions with other molecules.
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Tₘ (Melting Temperature)
Tₘ, or melting temperature, is the temperature at which half of the DNA strands are in the double-helix state and half are in the denatured single-strand state. It is a critical parameter that reflects the stability of the DNA molecule, influenced by factors such as base composition, length, and the presence of salts. Understanding Tₘ is vital for applications like PCR, where precise temperature control is necessary for successful amplification of DNA.
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